Immunological combination compositions and methods

ABSTRACT

Immununological compositions and methods for making and using them. The compositions contain at least one antigen and at least one lipoprotein and optionally an adjuvant. The lipoprotein can itself be antigenic or immunogenic. The antigen can be influenza HA and the lipoprotein a recombinantly expressed product having an OspA leader for lipidation and PspA for the protein portion. The antigen can be OspC and the lipoprotein OspA. The components of the composition are co-administered. A potentiated immunological response is obtained by the compositions and methods.

REFERENCE TO RELATED APPLICATIONS

This application is divisional of application Ser. No. 10/096,687, filedMar. 12, 2002. now U.S. Pat. No. 6,984,385, which is a continuation ofapplication Serial No. 08/588,621, filed Jan. 19, 1996, now U.S. Pat.No. 6,379,675, which is a continuation-in-part of application Ser. No.08/476,656, filed Jun. 7, 1995, now U.S. Pat. No. 6,251,405.

Reference, especially wit respect to recombinant Borrelia proteins, ismade to each of applications Ser. No. 07/973,338, filed Oct. 29, 1992(abandoned); Ser. No. 08/373,455 (Rule 62 FWC of U.S. Ser. No.07/973,338), filed Jan. 17, 1995 (abandoned); Ser. No. 07/888,765, filedMay 27, 1992 (abandoned); Ser. No. 08/211,891, filed Oct. 16, 1992(abandoned: national phase of PCT/US92/08697); and Ser. No. 07/779,048,flIed Oct. 18, 1991 (abandoned). Reference, especially with respect tostructural genes of pneumococcal proteins, epitopic regions thereof, andadministration of pneumococcal proteins, is made to each of applicationsSer. No. 07/656,773, filed Feb. 15, 1991 (abandoned); Ser. No. 07/835698, filed Feb. 12, 1992 (abandoned) Ser. No. 08/072,065, filed Jun. 3,1993 (abandoned); Ser. No. 08/072,068, filed Jun. 3, 1993 (abandoned);Ser. No. 08/214,222 filed Mar. 17,1994. now U.S. Pat. No. 5,804,193;Ser. No. 08/214,164, filed Mar. 17, 1994, now U.S. Pat. No. 5,728,387;Ser. No. 08/247,491, filed May 23, 1994. now U.S. Pat. No. 5,965,400;Ser. No. 08/048,896, filed Apr. 20, 1993 (abandoned); Ser. No.08/246,636, filed May 20, 1994. now U.S. Pat. No. 5,965,141; Ser. No.08/458,399 (continuation-in-part of application Ser. No. 08/246,636.filed Oct. 7, 1994. now US. Pat. No. 5,965,141) filed Jun. 2, 1995. nowU.S. Pat. No. 6,231,870; Ser. No. 08/446,201 filed May 19, 1995, nowU.S. Pat. No. 6,042,838; Ser. No. 08/312,949, filed Sep. 30, 1994. nowUS. Pat. No. 6,027,734. With respect to Expression of Lipoproteins,reference is made to application Ser. No. 08/475,781, filed Jun. 7, 1995(abandoned). And, with respect to Compostions and Methods forAdministering Borrelia Burgdorferi Antigens mucosally, e.g., orally, forsimulating an immunological response, reference is made to Barbour etal., application Ser. No. 08/588,637, filed Jan. 19, 1996, now U.S. Pat.No. 7,094,391.

Each of the aforementioned applications is hereby incorporated herein byreference. Several documents are cited in the following text, and eachis also hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions for eliciting animmunological response in a host, animal or human, and methods formaking and using the same. The invention further relates to suchcompositions and methods wherein the composition comprises an antigenand a lipoprotein adsorbed to an adjuvant. More preferably, thelipoprotein is also antigenic or immunogenic, and thus the compositioncan be a combination, multivalent or “cocktail” composition.Accordingly, the invention also relates to co-administration of at leastone antigen and at least one lipoprotein in a composition which caninclude additional ingredients, such as an adjuvant.

The lipoprotein can be a naturally occurring lipoprotein or arecombinant lipoprotein. The recombinant lipoprotein can be fromexpression by a vector of homologous sequences for the lipidated andprotein portions of the lipoprotein, i.e., the sequences for thelipidation and protein can naturally occur together. In such arecombinant lipoprotein, the lipidation thereof can be from expressionof a first nucleic acid sequence and the protein thereof can be fromexpression of a second nucleic acid sequence, wherein the first andsecond nucleic acid sequences, which do not naturally occur together,and such sequences can be expressed as a contiguous lipoprotein. Thus,the invention relates to compositions and methods involvingadministration of lipoproteins, including recombinant lipoproteins; andthe recombinant lipoproteins can be similar to native proteins, or novelhybrid proteins.

The invention further relates to the aforementioned compositions foreliciting an immunological response and methods for making and using thesame wherein the lipoprotein is recombinantly expressed lipoprotein fromexpression of such aforementioned first and second nucleic acidsequences wherein the first nucleic acid sequence encodes a Borrelialipoprotein leader sequence; preferably such a recombinant lipidatedprotein expressed using the nucleic acid sequence encoding the OspAleader sequence. In a preferred embodiment the lipoprotein can be OspA;and thus, the invention also relates to recombinant OspA and usesthereof the compositions and methods.

Several publications are referenced in this application. Full citationto these references is found at the end of the specification immediatelypreceding the claims or where the publication is mentioned; and each ofthese publications is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Immunogenicity can be significantly improved if an antigen isco-administered with an adjuvant, commonly used as 0.001% to 50%solution in phosphate buffered saline (PBS). Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune response to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccarides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune response.Aluminum hydroxide and aluminum phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and,more recently, a HBsAg vaccine has been adjuvanted with alum.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramoyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes. Toefficiently indvice humoral immune response (HIR) and cell-mediatedimmunity (CMI), immunogens are preferably emulsified in adjuvants.

Desirable characteristics of ideal adjuvants include any or all of:

-   -   (1) lack of toxicity;    -   (2) ability to stimulate a long-lasting immune response;    -   (3) simplicity of manufacture and stability in long-term        storage;    -   (4) ability to elicit both CMI and HIR to antigens (5)        administered by various routes;    -   (5) synergy with other adjuvants;    -   (6) capability of selectively interacting with populations of        antigen presenting cells (APC);    -   (7) ability to specifically elicit appropriate T_(H)1 or T_(H)2        cell-specific immune responses; and    -   (8) ability to selectively increase appropriate antibody isotype        levels (for example IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al. on Aug. 8, 1989 whichis incorporated herein by reference thereto teaches glycolipid analogsincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmune-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No.4,855,283) reported that N-glycolipids analogs displaying structuralsimilarities to the naturally occurring glycolipids, such asglycosphingolipids and glycoglycerolipids, are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain alkylamines and fatty acids that are linked directly with thesugar through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, assigned to ConnaughtLaboratories Limited and incorporated herein by reference thereto,teaches that octadecyl tyrosine hydrochloride (OTH) functions as anadjuvant when complexed with tetanus toxoid and formalin inactivatedtype I, II and III poliomyelitis virus vaccine. Octodecyl esters ofaromatic amino acids complexed with a recombinant hepatitis B surfaceantigen, enhanced the host immune responses against hepatitis B virus.

Bessler et al., “Synthetic lipopeptides as novel adjuvants,” in the 44thForum In Immunology (1992) at page 548 et seq., especially at 548–550,incorporated herein by reference, is directed to employing lipopeptidesas adjuvants when given in combination with an antigen. The lipopeptidestypically had P3C as the lipidated moiety and up to only 5 amino acids,e.g., P3C-SG, P3C-SK4, P3C-SS, P3C-SSNA, P3C-SSNA. The lipopeptide wascoupled with or added to only certain antigens or to non-immunogenicproteins, such as P3C-SSNA supplementing S. typhimurium vaccine, PC3-SScoupled to VP1(135–154) of foot-and-mouth disease, PC3-SG-OSu coupled tonon-immunogenic protein hirudin, P3C-SK coupled to FITC or DNP or P3C-SGcoupled to a metabolite from Streptomyces venezuelae. While adjuvantmixing and conjugating procedures of Bessler can be employed in thepractice of the present invention, Bessler fails to teach or suggestemploying a lipoprotein with at least one antigen in a composition,especially such compositions wherein the lipoprotein is also antigenic,or the immunological combination compositions and methods of thisinvention.

In this regard, a distinction between a peptide, especially a peptidehaving up to only about 5 amino acids, and a protein is being made, asis a distinction between an antigenic lipoprotein and a non-antigeniclipopeptide, inter alia. Peptides differ immunologically from proteinsin that short peptides have the potential for direct presentation by themajor histocompatibility complex (MHC), while proteins requireprocessing prior to presentation to T-cells. A peptide further differsfrom a protein in that a protein is large enough that it is capable offorming functional domains (i.e., having tertiary structure), whereas apeptide cannot.

Nardelli et al. [Vaccine (1994), 12(14):1335–1339] covalently linked atetravalent multiple antigen peptide containing a gp120 sequence to alipid moiety and orally administered the resulting synthetic lipopeptideto mice. It was found that both mucosal IgA response and systemic plasmaIgG were stimulated, and cell-mediated immunity, as shown by lymphokineproduction and generation of a specific cytotoxic response, was induced.Only a short peptide was used, rather than a whole lipoprotein, andthere is no teaching or suggestion that the synthetic lipopeptide couldbe used as an adjuvant for other proteins. In fact, this referenceactually teaches away from the use of lipoproteins, which are moresoluble than lipopeptides, as immunogens; see, e.g. p. 1338, last line(“soluble proteins are not immunogens by oral routes”).

Croft et al. [J. Immunol. (1991), 146(5): 793–796] have covalentlycoupled integral membrane proteins (Imps) isolated from E. coli tovarious antigens and obtained enhanced immune responses by intramuscularinjection into mice and rabbits. However, there are disadvantages tocoupling the lipoprotein and the antigen covalently. Important epitopesmay be damaged, and the coupling procedure is difficult to control andoften requires the use of toxic cross-linkers. Thus, it would beadvantageous to provide a method for inducing an enhanced immunologicalresponse which does not require that the antigen be cross-linked to aprotein. Moreover, when the antigen CSP-OVA was merely mixed, ratherthan covalently linked, with the lipoprotein TraT, only a small increasein antibody response was obtained. Croft et al. therefore concluded thatthe lipid is not necessary for the adjuvant effect, contrary to thesurprising findings of the present inventors.

U.S. Pat. No. 4,439,425 relates to lipopeptides having 2 to 10 aminoacids and their prophylactic administration by oral or rectal routes.

Bessler et al. [“Synthetic Lipopeptide Conjugates Constitute EfficientNovel Immunogens and Adjuvants in Parenteral and Oral Immunization”(Abstract), Meeting on Molecular Approaches to the Control of InfectiousDiseases, (Sep. 13–17, 1995), Cold Spring, Harbor Laboratory (not priorart in view of Jun. 7, 1995 filing date of U.S. Ser. No. 08/476,656)]relates to the oral administration of lipopeptides having six aminoacids which were covalently coupled to antigens. The lipopeptide-antigenconjugates were found to induce a hapten-specific immune response.

Schlecht et al. [Zbl. Bakt. (1989) 271:493–500] relates to Salmonellatyphimurium vaccines supplemented with synthetically preparedderivatives of a bacterial lipoprotein having five amino acids. Thevaccines were administered by two intraperitoneal injections andchallenged intraperitoneally with graded doses of S. typhimurium. Whenthe protective capacity of the supplemented vaccines was compared withthat of the unsupplemented vaccine, it was found that 90% of the S.typhimurium vaccine could be replaced by the lipopeptide without arecognizable decrease in protective capacity.

Substantial effort has been directed toward the development of a vaccinefor Lyme disease. Two distinct approaches have been used for vaccinedevelopment. One approach is to use a vaccine composed of wholeinactivated spirochetes, as described by Johnson in U.S. Pat. No.4,721,617. A whole inactivated vaccine has been shown to protecthamsters from challenge and has been licensed for use in dogs.

Due to the concerns about cross-reactive antigens within a whole cellpreparation, human vaccine research has focused on the identificationand development of non-cross-reactive protective antigens expressed byB. burgdorferi. Several candidate antigens have been identified to date.Much of this effort has focused on the most abundant outer surfaceprotein of B. burgdorferi, namely outer surface protein A (OspA), asdescribed in published PCT patent application WO 92/14488, assigned tothe assignee hereof. Several versions of this protein have been shown toinduce protective immunity in mouse, hamster and dog challenge studies.Clinical trials in humans have shown the formulations of OspA to be safeand immunogenic in humans [Keller et al., JAMA (1994) 271:1764–1768].Indeed, one formulation containing recombinant lipidated OspA asdescribed in the aforementioned WO 92/14488, is now undergoing Phase IIIsafety/efficacy trials in humans.

While OspA is expressed in the vast majority of clinical isolates of B.burgdorferi from North America, a different picture has emerged fromexamination of the clinical Borrelia isolates in Europe. In Europe, Lymedisease is caused by three genospecies of Borrelia, namely B.burgdorferi, B. garinii and B. afzelli. In approximately half of theEuropean isolates, OspA is not the most abundant outer surface protein.A second outer surface protein C (OspC) is the major surface antigenfound on these spirochetes. In fact, a number of European clinicalisolates that do not express OspA have been identified. Immunization ofgerbils and mice with purified recombinant OspC produces protectiveimmunity to B. burgdorferi strains expressing the homologous OspCprotein [V. Preac-Mursic et al., INFECTION (1992) 20:342–349; W. S.Probert et al., INFECTION AND IMMUNITY (1994) 62:1920–1926]. The OspCprotein is currently being considered as a possible component of asecond generation Lyme vaccine formulation.

Recombinant proteins are promising vaccine or immunogenic compositioncandidates, because they can be produced at high yield and purity andmanipulated to maximize desirable activities and minimize undesirableones. However, because they can be poorly immunogenic, methods toenhance the immune response to recombinant proteins are important in thedevelopment of vaccines or immunogenic compositions. Moreover, it wouldbe greatly desired to be able to administer such proteins in combinationwith other antigens.

A very promising immune stimulator is the lipid moietyN-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propyl-cysteine, abbreviatedPam₃Cys. This moiety is found at the amino terminus of the bacteriallipoproteins which are synthesized with a signal sequence that specifieslipid attachment and cleavage by signal peptidase II. Synthetic peptidesthat by themselves are not immunogenic induce a strong antibody responsewhen covalently coupled to PamjCys [Bessler et al. (1992)].

In addition to an antibody response, one often needs to induce acellular immune response, particularly cytoxic T lymphocytes (CTLs).Pam₃Cys-coupled synthetic peptides are extremely potent inducers ofCTLs, but no one has yet reported CTL induction by large recombinantlipoproteins.

The nucleic acid sequence and encoded amino acid sequence for OspA areknown for several B. burgdorferi clinical isolates and is described, forexample, in published PCT application WO 90/04411 (Symbicom AB) for B31strain of B. burgdorferi and in Johnson et al., Infect. Immun.60:1845–1853 for a comparison of the ospA operons of three B.burgdorferi isolates of different geographic origins, namely B31, ACA1and Ip90.

As described in WO 90/04411, an analysis of the DNA sequence for the B31strain shows that the OspA is encoded by an open reading frame of 819nucleotides starting at position 151 of the DNA sequence and terminatingat position 970 of the DNA sequence (see FIG. 1 therein). The firstsixteen amino acid residues of OspA constitute a hydrophobic signalsequence of OspA. The primary translation product of the full length B.burgdorferl gene contains a hydrophobic N-terminal signal sequence whichis a substrate for the attachment of a diacyl glycerol to the sulfhydrylside chain of the adjacent cysteine residue. Following this attachment,cleavage by signal peptidase II and the attachment of a third fatty acidto the N-terminus occurs. The complete lipid moiety is termed Pam₃Cys.It has been shown that lipidation of OspA is necessary forimmunogenicity, since OspA lipoprotein with an N-terminal Pam₃Cys moietystimulated a strong antibody response, while OspA lacking the attachedlipid did not induce any detectable antibodies [Erdile et al., Infect.Immun., (1993), 61:81–90].

Published international patent application WO 91/09870 (MikrogenMolekularbiologische Entwicklungs-GmbH) describes the DNA sequence ofthe ospC gene of B. burgdocferi strain Pko and the OspC (termed pC inthis reference) protein encoded thereby of 22 kDa molecular weight. Thissequence reveals that OspC is a lipoprotein that employs a signalsequence similar to that used for OspA. Based on the findings regardingOspA. one might expect that lipidation of recombinant OspC would beuseful to enhance its immunogenicity; but, as discussed inabove-referenced application Ser. No. 08/475,781, filed Jun. 7, 1995(abandoned; the continuation of which is application Ser. No.09/067,453, filed Apr. 28, 1998 (now U.S. Pat. No. 6,538,118 B1)), thetherein applicants experienced difficulties in obtaining detectableexpression of recombinant OspC. It would be useful to enhance theimmunogenicity of recombinant OspC. Moreover, it would be useful to havea multivalent Lyme Disease immunological composition which containsantigens against both North American and European Borrelia isolates.

Streptoccus pneumoniae causes more fatal infections world-wide thanalmost any other pathogen. In the U.S.A., deaths caused by S. pneumoniaerival in numbers those caused by AIDS. Most fatal pneumoccal infectionsin the U.S.A. occur in individuals over 65 years of age, in whom S.pneumoniae is the most common cause of community-acquired pneumonia. Inthe developed world, most pneumococcal deaths occur in the elderly, orin immunodeficient patents including those with sickle cell disease. Inthe less-developed areas of the world, pneumococcal infection is one ofthe largest causes of death among children less than 5 years of age. Theincrease in the frequency of multiple antibiotic resistance amongpneumococci and the prohibitive cost of drug treatment in poor countriesmake the present prospect for control of pneumococcal diseaseproblematical.

The reservoir of pneumococci that infect man is maintained primarily vianasopharyngeal human carriage. Humans acquire pneumococci first throughaerosols or by direct contact. Pneumococci first colonize the upperairways and can remain in nasal mucosa for weeks or months. As many as50% or more of young children and the elderly are colonized. In mostcases, this colonization results in no apparent infection. In someindividuals, however, the organism carried in the nasopharynx can giverise to symptomatic sinusitis of middle ear infection. If pneumococciare aspirated into the lung, especially with food particles or mucus,they can cause pneumonia. Infections at these sites generally shed somepneumococci into the blood where they can lead to sepsis, especially ifthey continue to be shed in large numbers from the original focus ofinfection. Pneumococci in the blood can reach the brain where they cancause meningitis. Although pneumococcal meningitis is less common thanother infections caused by these bacteria, it is particularlydevastating; some 10% of patients die and greater than 50% of theremainder have life-long neurological sequelae.

In elderly adults, the present 23-valent capsular polysaccharide vaccineis about 60% effective against invasive pneumococcal disease withstrains of the capsular types included in the vaccine. The 23-valentvaccine is not effective in children less than 2 years of age because oftheir inability to make adequate responses to most polysaccharides.Improved vaccines that can protect children and adults against invasiveinfections with pneumococci would help reduce some of the mostdeleterious aspects of this disease.

The S. pneumoniae cell surface protein PspA has been demonstrated to bea virulence factor and a protective antigen. In published internationalpatent application WO 92/14488, there are described the DNA sequencesfor the pspA gene from S. pneumoniae R×l , the production of a truncatedform of PspA by genetic engineering, and the demonstration that suchtruncated form of PspA confers protection in mice to challenge with livepneumococci.

In an effort to develop a vaccine or immunogenic composition based onPspA, PspA has been recombinantly expressed in E. coli. It has beenfound that in order to efficiently express PspA, it is useful totruncate the mature PspA molecule of the Rxl strain from its normallength of 589 amino acids to that of 314 amino acids comprising aminoacids 1 to 314. This region of the PspA molecule contains most, if notall, of the protective epitopes of PspA. However, immunogenicity andprotection studies in mice have demonstrated that the truncatedrecombinant form of FspA is not immunogenic in naive mice. Thus, itwould be useful to improve the immunogenicity of recombinant PspA andfragments thereof. Moreover, it would be highly desirable to employ apneumococcal antigen in a combination or multivalent composition. Forinstance, influenza (Flu) is a problematical infection, especially inthe elderly and the young, as well as pneumonia; and, yearly Flu shotsare common, especially in North America. Thus, it would be desirable tobe able to administer Flu and pneumococcal antigens in one preparation.

Helicobacter pylori is the spiral bacterium which selectively coloniizeshuman gastric mucin-secreting cells and is the causative agent in mostcases of nonerosive gastritis in humans. Recent research activityindicates that H. pylori, which has a high urease activity, isresponsible for most peptic ulcers as well as many gastric cancers. Manystudies have suggested that urease, a complex of the products of theureA and ureB genes, may be a protective antigen. However, until now ithas not been known how to produce a sufficient mucosal immune responseto urease without cholera toxin or related adjuvants.

Antigens or immunogenic fragments thereof stimulate an immune responsewhen administered to a host. Such antigens, especially whenrecombinantly produced, may elicit a stronger response when administeredin conjunction with adjuvant. Currently, alum is the only adjuvantlicensed for human use, although hundreds of experimental adjuvants suchas cholera toxin B are being tested. However, these adjuvants havedeficiencies. For instance, while cholera toxin is a good adjuvant, itis highly toxic. On the other hand, cholera toxin B, while non-toxic,has no adjuvant activity. It would thus be desirable to provideimmunological compositions capable of eliciting a strong responsewithout the need for an adjuvant.

In certain instances when multiple antigens (two or more) areadministered in the same preparation or sequentially, a phenomenoncalled efficacy interference occurs. Simply, due to the interaction ofone or more antigens in the preparation with the host immunologicalsystem, the second or other antigens in the preparation fail to elicit asufficient response, i.e., the efficacy of the latter antigen(s) isinterfered with by the former antigen(s). It would thus be desirable toprovide multivalent immunological compositions which do not give rise tothis efficacy interference phenomenon; for instance, without wishing tonecessarily be bound by any one particular theory, because the secondantigen is a lipoprotein and as such is having an adjuvanting effect onthe first antigen and, when in a combination composition with anadjuvant, a synergistic potentiating effect is obtained (whereby thefirst antigen is not interfering with the second antigen and viceversa).

More generally it would be desirable to enhance the immunogenicity ofantigens by methods other than the use of an adjuvant, and to have theability to employ such a means for enhanced immunogenicity with anadjuvant, so as to obtain an even greater immunological response.

Above-referenced application Ser No. 08/446,201 (now U.S. Pat No.6,042,838 discloses that mucosal administration of killed wholepneurnococci, lysate of pneumococci or isolated and purified PapA, aswell as immunogenic fragments thereof, particularly when administeredwith an adjuvant, provides protection in animals against pneumococcalcolonization and systemic infection. It has now been surprisingly foundthat mucosal administration of other antigens, such as urease, alongwith a lipoprotein, elicits systemic and local responses in animalswithout the use of an adjuvant.

It is believed that heretofore the art has not taught or suggested:immunological compositions comprising at least one antigen and alipoprotein, and, optionally, an adjuvant, more preferably an antigen,an antigenic lipoprotein and, optionally, an adjuvant, and methods foradministering the same as a multivalent composition, or foradministering those components simultaneously or sequentially,especially such compositions and methods having enhanced immunogenicity.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide immunological compositionsand methods for making and using the same.

It is a further object of the invention to provide immunologicalcompositions having enhanced immunogenicity; or, compositions theadministration of which potentiates the immunological response.

It is another object of the invention to provide methods for inducing animmunological response, preferably a potentiated response, involvingadministration to a suitable host such immunological compositions.

It is yet an additional object of the invention to provide animmunological composition comprising at least one antigen and at leastone lipoprotein and, optionally, an adjuvant, preferably suchcompositions wherein the lipoprotein is antigenic.

It is still a further object of the invention to provide a method forinducing or potentiating an immunological response comprisingadministering to a host, animal or human, at least one antigen and atleast one lipoprotein, and optionally, an adjuvant; and more preferablysuch methods wherein the lipoprotein is antigenic.

It has surprisingly been found that administration to a host of at leastone lipoprotein with at least one antigen provides an immunologicalresponse by the host. The imrounological response is generally betterthan that obtained by administration of the antigen alone.

Moreover, it has also surprisingly been found that administration to ahost of at least one antigen, at least one lipoprotein and, optionallyan adjuvant by either co-administration or by sequential administration(over a suitable time period such that each of the antigen, adjuvant andlipoprotein are present within the host at the same time) obtains animmunological response to the antigen by the host. This immunologicalresponse is generally better than that obtained by administration of theantigen alone or by administration of the antigen and adjuvant.Lipidated proteins appear to stimulate the immune response, in themanner of the adjuvant cholera toxin B.

Furthermore, it has additionally been surprisingly found that in theseadministrations the lipoprotein itself can be immunogenic or antigenic,e.g., be an antigen, and that not only is the immunological response tothe antigen by the host obtained; but also, an immunological response tothe antigenic lipoprotein is obtained. The immunological response to theantigenic lipoprotein can be as good as, or better than, that obtainedby administration of the lipoprotein alone or with an adjuvant; and, theimmunological response to the antigen can be better than that obtainedby administering the antigen alone or the antigen and adjuvant.

The term lipoprotein as used herein is meant to exclude prior artlipopeptides; ergo, a lipoprotein can have more than 2 to 10 aminoacids, or more than 18 to 20 amino acids, or greater than 24 aminoacids, or 30 or more amino acids. Lipoproteins are larger moleculeswhich reduce the amount of antigen and/or administrations of theantigen, despite a prejudice in the art against lipoproteins, e.g.,Vaccine (1994) 12(14):1335, 1338 last line, column 1, to first line,column 2 (“soluble proteins . . . not immunogenic [by oral routes]”).Prior lipopeptides, due to their small size, can have at most oneepitope whereas lipoproteins that can be used in the present inventioncan have more than one epitope, e.g. one B and one T, or can even beantigenic in their own right. Lipopeptides, in addition to being shorterand having less molecular weight than lipoproteins, and being difficultto synthesize because usually are made by Merrifield or other synthesismethods, differ from lipoproteins in that lipoproteins are larger,generally not made by Merrifield synthesis methods, and can be fromisolation from natural sources or from recombinant techniques. That is,lipopeptides of the prior art were synthetically made, which limitstheir size to no more than about thirty amino acids. Lipoproteins arelarger and of greater molecular weight than lipopeptides, and, unlikelipopeptides, are generally not made by Merrifield synthesis methods.Lipoproteins can be isolated from natural sources or produced byrecombinant techniques. Further, lipoproteins are more soluble thanlipopeptides. Additionally, peptides do not have quaternary or tertiarystructure whereas proteins can have quaternary and/or tertiarystructure. Based upon their ability to form tertiary structure, proteinshave the ability to form functional domains which peptides cannot. Thus,there are several differences between prior “lipopeptides” and“lipoproteins” as used in this invention.

The lipoproteins formulations of the invention can be administerednasally and this is advantageous.

According to the present invention, it also has been found that alipoprotein administered with an antigen according to the presentinvention is 500 times more potent then administration of a lipopeptideand an antigen.

Accordingly, the present invention provides an immunological compositioncomprising at least one antigen and at least one lipoprotein. Thecomposition can further optionally, but not necessarily, comprise anadjuvant. Preferably the lipoprotein is an antigen. The immunologicalcomposition can be a vaccine.

The present invention further comprises a method for inducing animmunological response in a host comprising administering theaforementioned immunological composition. The method can be for inducinga protective response, e.g., when the immunological composition is avaccine.

The present invention further comprises a method for inducing animmunological response comprising sequentially administering a firstcomposition comprising an antigen, and a second composition comprising alipoprotein. Optionally either the first or second composition, or boththe first and second compositions can further comprise an adjuvant.Preferably the lipoprotein is an antigen. The sequential administrationshould be undertaken over a suitable period of time whereby each of theantigen, lipoprotein and optional adjuvant is present at the same timein the host; and, such a time period can be determined by the skilledartisan, from this disclosure, without undue experimentation and bymethods within the ambit of the skilled artisan, such as host seratitrations involving analysis thereof for the presence of antigen orantibody by, for instance, ELISA analysis. The administration may bemucosal, e.g., intragastric or intranasal.

The present invention particularly involves methods for inducing animmunological response in a host comprising the steps of mucosallyadministering to the host at least one antigen, and mucosallyadministering to the host at least one lipoprotein. The administrationcan be simultaneous or sequential. The antigen may be a bacterialprotein or fragment thereof, e.g. urease.

The “antigen” in the inventive compositions and methods can be anyantigen to which one wishes to elicit an immunological response in ahost, animal or human. For instance, without wishing to necessarilylimit the invention, the antigen can be: a Borrelia antigen, e.g., OspA,OspC, OspB, OspD; a pneumococcal antigen, e.g., PspA; an influenza (Flu)antigen such as HA; a pertussis or whooping cough antigen such as thepertussis 69 KD polypeptide; a hepatitis antigen, e.g., hepatitis Bantigen such as hepatitis B surface antigen; a Helicobacter pyloriantigen such as urease; a rabies virus antigen, e.g., rabies G antigen;a flavivirus antigen, e.g., a Japanese encephalitis virus, Dengue virusor yellow fever virus antigen; a chicken pox virus antigen; a diphtheriaantigen; a C. tetani antigen, e.g., tetanus toxoid; a mumps virusantigen; a measles virus antigen; a malaria antigen; a herpes virusantigen, such as an alphaherpesvirus, betaherpesvirus orgammaherpesvirus antigen, e.g., a herpes virus glycoprotein, forinstance an equine herpesvirus antigen, e.g., gp13, gp14, gD, gp63, orgE, a pseudorabies virus antigen, e.g., gp50, gpII, gpIII, gpI, aherpessimplex virus antigen, e.g., gC, gD, a bovine herpes virusantigen, e.g., gl , a feline herpes virus antigen, e.g., gB, anEpstein-Barr virus antigen, e.g., gp220, gp340, or gH, or a humancytomegalovirus antigen, e.g., gB; a human immunodeficiency virusantigen, e.g., gp160 or gp120; a simian immunodeficiency virus antigen;a bovine viral diarrhea virus antigen; an equine influenza virusantigen; a feline leukemia virus antigen; a canine distemper virusantigen, e.g., HA or F glycoproteins; a canine adenovirus antigen, e.g.,canine adenovirus type 2 antigen; a canine coronavirus antigen; a canineparainfluenza antigen; a canine parvovirus antigen; a Hantaan virusantigen; an avian influenza virus antigen e.g., a nucleoprotein antigen;a Newcastle Disease virus antigen, e.g., F, HN; an antigen of rousassociated virus, e.g., an RAV-1 envelope antigen; an infectiousbronchitis virus antigen, e.g., a matrix antigen or a preplomer antigen;an infectious bursal disease virus antigen; a cholera antigen; a tumorassociated antigen; a feline immunodeficiency virus antigen; afoot-and-mouth disease virus antigen; a Marek's Disease Virus antigen; aStaphylococci antigen; a Streptococci antigen; a Haemophilus influenzaantigen, e.g., group b polysaccharide-protein conjugates; a papillomavirus; a poliovirus antigen; a rubella virus antigen; a poxvirus, suchas smallpox antigen, e.g., vaccinia; a typhus virus antigen; a typhoidvirus antigen; a tuberculosis virus antigen; an HTLV antigen; or, otherbacteria, virus or pathogen antigen, such as a bacterial or viralsurface antigen or coat protein.

The antigen can be a known antigen; can be isolated from the bacteria,virus or pathogen; or, can be a recombinant antigen from expression ofsuitable nucleic acid coding therefor by a suitable vector, andisolation and/or purification of the recombinant antigen. The selectionof the antigen is, of course, dependent upon the immunological responsedesired and the host.

The lipoprotein can be any lipoprotein which is compatiblephysiologically with the host. Most preferably it is a bacteriallipoprotein or a lipoprotein having a bacterial lipid moiety.

The lipoprotein is preferably itself also an antigen. Thus, thelipoprotein is preferably an outer membrane component of a pathogen,e.g., virus or bacteria, more preferably a lipoprotein which has anextrinsic or peripheral protein such that the lipoprotein is extractedwith mild conditions or detergent without substantial denaturation orloss of lipid moiety (so as to retain epitopes). However, any antigeniclipoprotein can be employed in the practice of the invention. And, thelipoprotein can be isolated from a suitable physiological source, orfrom an organism, e.g., bacteria; or can be recombinantly produced.Thus, the lipidated Borrelia antigens, e.g., recombinant OspA, and, thelipidated OspA and Borrelia fractions containing lipidated proteins(isolated by mild conditions) disclosed in the applications referencedin the Reference to Related Applications, and in WO 90/04411(incorporated herein by reference) can be used as the lipoprotein in thepractice of the invention. Of course, the “antigen” and the“lipoprotein” in the invention are separate, different ingredients (suchthat, for instance, when the “lipoprotein” is OspA, it is not also the“antigen”).

In application Ser. No. 08/475,781 filed Jun. 7, 1995 (abandoned; thecontinuation of which is application Ser. No. 09/067,453, filed Apr. 28,1998 (now U.S. Pat No. 6,538,118 B1)) and incorporated herein byreference, recombinant lipoproteins, especially antigenic recombinantlipoproteins, for instance, those from expression of the leader sequenceof OspA for the lipidation thereof, are disclosed; and, thoserecombinant lipoproteixis may be employed in the practice of theinvention. As to expression of recombinant proteins, it is expected thatthe skilled artisan is familiar with the various vector systemsavailable for such expression, e.g., bacteria such as E. coli andbacterial viruses, and the like.

The adjuvant can be any vehicle which would typically enhance theantigenicity of the antigen, e.g., a suspension or gel of minerals (forinstance, alum, aluminum hydroxide or phosphate) on which the antigen isadsorbed; or a water-in-oil emulsion in which antigen solution isemulsified in mineral oil (e.g., Freund's incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (e.g., Freund'scomplete adjuvant); or cholera toxin (sometimes with cholera toxin B,which may enhance the effect); or, any of the other adjuvants known inthe art, or discussed in the Background of the Invention. The antigenand/or the lipoprotein can be absorbed onto or coupled with theadjuvant.

Presently preferred embodiments of the invention involve: alum as theadjuvant if an adjuvant is present; OspA, or a recombinant OspAleader/PspA, a recombinant OspA leader/OspC, a recombinant OspAleader/UreA of H. pylori, or, a recombinant OspA leader/UreB of H.Pylori as the lipoprotein (OspA leader/PspA is a recombinant lipoproteinhaving a lipidated moiety from expression of the OspA leader nucleicacid sequence and a protein moiety from expression of a pspA nucleicacid sequence; OspA leader/OspC is analogous to OspA leader/PspA, exceptthat the protein moiety is from expression of an ospC nucleic acidsequence and OspA leader/ureA and OspA leader/ureB are also analogous toOspA leader/PspA, except that the protein moiety is from expression of aureA or ureB nucleic acid sequence); and OspC or another Borreliaantigen, or an influenza antigen, e.g., HA (such as from influenza A,e.g., Texas strain), or urease as the antigen. Particular embodimentscan include compositions: (i) comprising alum [adjuvant], OspA[lipoprotein] and another Borrelia antigen such as OspC [antigen]; (ii)comprising alum [adjuvant], OspA [antigen], and OspA leader/OspC[lipoprotein]; (iii) comprising alum [adjuvant], OspA leader/PspA[lipoprotein] and influenza antigen, e.g., influenza A HA [antigen] (iv)OspA [lipoprotein} and an H. pylori antigen, e.g., urease [antigen].

Other objects and embodiments of the invention are disclosed in or areobvious variants from the following description.

BRIEF DESCRIPTION OP THE DRAWINGS

In the following detailed description, reference is made to theaccompanying drawings, wherein:

FIG. 1 is a graphical representation of the immune response of miceimmunized with OspC formulations with or without purified lipidated OspAand with or without alum as an adjuvant as measured in an anti-OspCELISA at day 63 after immunization; and

FIG. 2 is a graphical representation of the immune response of miceimmunized with OspC formulations with or without purified lipidated OspAand with or without alum as an adjuvant as measured in an anti-OspCELISA at day 91 after immunization.

FIG. 3 is a graphical representation of the immune response of miceimmunizing twice, intranasally, with either lipidated or non-lipidatedOspA as measured in an anti-OspA ELISA at day 9 after the secondimmunization.

FIG. 4 is a graphical representation of the immune response of miceimmunized twice, both intranasally and intragastrically, with eitherjack bean urease alone or both urease and OspA, as measured in ananti-urease ELISA at day 9 after the second immunization.

FIG. 5 is a graphical representation of the immune response of miceimmunized twice, intranasally, with jack bean urease, either above orwith OspA or cholera toxin, as measured in an anti-urease ELISA at day 9after the second immunization.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the invention involves immunological compositionsand methods for making and using (e.g., administering) then which, in abroad sense, include immunological compositions comprising an antigenand a lipoprotein and optionally including an adjuvant; and the methodsbroadly include administering such compositions to a suitable host suchthat there is co-administration of the antigen and lipoprotein andoptional adjuvant, or sequentially administering the components thereof.

It has now surprisingly been found that mucosal administration of anantigen, e.g., a bacterial protein or fragment thereof, and alipoprotein produces both local and serum immune responses. Theprincipal determinant of specific immunity at mucosal surfaces issecretory IgA (S-IgA) which is physiologically and functionally separatefrom the components of the circulatory immune system. S-IgA antibodyresponses may be induced locally by the application of suitableimmunogens to a particular mucosal site. The bulk of mucosal S-IgAresponses, however, are the results of immunity generated via the commonmucosal immune system (CMIS) [Mestecky, J. J. Clin Immunol. (1987)7:265–276], in which immunogens are taken up by specializedlympho-epithelial structures, collectively referred to asmucosa-associated lymphoid tissue (MALT). The best studied immunologiclympho-epithelial structures are the gut-associated lymphoid tissues(GALT), such as intestinal Peyer's patches. It is now clear, however,that other structurally and functionally similar lymphoid folliclesoccur at other mucosal surfaces, including those of the respiratorytract [Croituru, K., et al., in “Handbook of Mucosal Immunology”(Bienenstock, J., ed.) San Diego, Calif.: Academic Press, Inc. (1994),141–149].

In the experimental results set forth in the Examples below, it is shownthat mice can be effectively immunized by intranasal (i.n.) orintragastric (i.g.) installation of bacterial protein immunogens inconjunction with a lipoprotein such as OspA. Specific IgA- andIgG-secreting cells are induced in the salivary glands and stomachs(stomachs not shown) and specific IgA antibodies are induced in saliva(not shown). Strong circulatory immune responses are also induced withIgG and IgA antibodies in the serum. Accordingly, it appears thatmucosal immunization with antigens along with lipoproteins is aneffective route for stimulating common mucosal responses as well ascirculatory antibody responses. Such immunization may be boththerapeutic and prophylactic.

The determination of the amount of antigen, lipoprotein and optionaladjuvant in the inventive compositions and the preparation of thosecompositions can be in accordance with standard techniques well known tothose skilled in the pharmaceutical or veterinary arts. In particular,the amount of antigen, lipoprotein and adjuvant in the inventivecompositions and the dosages administered are determined by techniqueswell known to those skilled in the medical or veterinary arts takinginto consideration such factors as the particular antigen, thelipoprotein, the adjuvant, the age, sex, weight, species and conditionof the particular patient, and the route of administration. Forinstance, dosages of particular antigens listed above for suitable hostsin which an immunelogical response is desired, are known to thoseskilled in the art, as is the amount of adjuvant typically administeredtherewith. Thus, the skilled artisan can readily determine the amount ofantigen and optional adjuvant in compositions and to be administered inmethods of the invention. Typically, an adjuvant is commonly used as0.001 to 50 wt % solution in phosphate buffered saline, and the antigenis present on the order of micrograms to milligrams, such as about0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % (see, e.g., the Examplesbelow).

The skilled artisan can refer to a known dosage for the particularantigen for a particular host to determine the amount of lipoprotein incompositions and administered in methods of the present invention, (ifthe lipoprotein is antigenic) such as the known dosages for OspA fromthe documents cited herein, or can scale the dosage for a particularhost from the documents cited herein and the Examples below (e.g., withrespect to OspA leader/PspA, OspA leader/Ospc, OspA leader/ureA, andOspA leader/ureB. Typically, however, the antigenic and/or recombinantlipoprotein is present in an amount on the order of micrograms tomilligrams, or, about 0.001 to about 20 wt %, preferably about 0.01 toabout 10 wt %, and most preferably about 0.05 to about 5 wt % (see,e.g., Examples below).

Of course, for any composition to be administered to an animal or human,including the components thereof, and for any particular method ofadministration, it is preferred to determine therefor: toxicity, such asby determining the lethal dose (LD) and LD₅₀ in a suitable animal modele.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable immunological response, such asby titrations of sera and analysis thereof for antibodies or antigens,e.g., by ELISA. Such determinations do not require undue experimentationfrom the knowledge of the skilled artisan, this disclosure and thedocuments cited herein. And, as discussed above, the time from forsequential administrations can be ascertained without undueexperimentation.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratorymucosa) etc., administration such as suspensions, syrups or elixirs;and, preparations for parenteral, subcutaneous, intradermal,intramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose or thelike. The compositions can also be lyophilized. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut.

If nasal or respiratory (mucosal) administration is desired,compositions may be prepared as inhalables, sprays and the like anddispensed by a squeeze spray dispenser, pump dispenser or aerosoldispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or,a dose having a particular particle size.

Compositions within the scope of this invention can contain a humectantto inhibit drying of the mucous membrane and to prevent irritation. Anyof a variety of pharmaceutically acceptable humectants can be employedincluding, for example sorbitol, propylene glycol or glycerol. As withthe thickeners, the concentration will vary with the selected agent,although the presence or absence of these agents, or theirconcentration, is not an essential feature of this invention.

Enhanced absorption across the mucosal and especially nasal membrane canbe accomplished employing a pharmaceutically acceptable surfactant.Typically useful surfactants for compositions include polyoxyethylenederivatives of fatty acid partial esters of sorbitol anhydrides such asTween 80, Polyoxynol 40 Stearate, Polyoxyethylene 50 Stearate andOctoxynol. The usual concentration is form 1% to 10% based on the totalweight.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, Parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like and will typicallycontain a sufficient amount of a thickening agent so that the viscosityis from about 2500 to 6500 cps, although more viscous compositions, evenup to 10,000 cps may be employed. Viscous compositions have a viscositypreferably of 2500 to 5000 cps, since above that range they become moredifficult to administer. However, above that range, the compositions canapproach solid or gelatin forms which are then easily administered as aswallowed pill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally, to animals, children, particularly small children,and others who may have difficulty swallowing a pill, tablet, capsule orthe like, or in multi-dose situations. Viscous compositions, on theother hand, can be formulated within the appropriate viscosity range toprovide longer contact periods with mucosa, such as the lining of thestomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form [e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form, or solid dosage form [e.g., whether the compositionis to be formulated into a pill, tablet, capsule, caplet, time releaseform or liquid-filled form].

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen,lipoprotein and optional adjuvant. Minor amounts of other ingredientssuch as pH adjusters (e.g., a base such as NaOH), emulsifiers ordispersing agents, buffering agents, preservatives, wetting agents,jelling agents, (e.g., methylcellulose), colors and/or flavors may alsobe present. The compositions can be isotonic, i.e., it can have the sameosmotic pressure as blood and lachrymal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to theantigen, lipoprotein and optional adjuvant. This will present no problemto those skilled in chemical and pharmaceutical principles, or problemscan be readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical andveterinary arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient or animal, and thecomposition form used for administration (e.g., solid vs. liquid).Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan, from this disclosure, thedocuments cited herein, the Examples below (e.g., from the Examplesinvolving mice), and the knowledge of antigens and lipoproteins andadjuvants herein mentioned.

The present invention also includes a method for inducing animmunological response in a host wherein the antigen and lipoprotein areadministered at one mucous membrane and a response is detectable atanother mucous membrane, e.g. nasal administration and vaginal response.This aspect of the invention is particularly useful for the treatment orprevention of sexually transmitted diseases.

Suitable regimes for initial administration and booster doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, the Examples below, and the knowledge ofantigens, lipoproteins and adjuvants herein mentioned without undueexperimentation.

The following Examples are provided for illustration and are not to beconsidered a limitation of the invention.

EXAMPLES Example 1

Construction of a pET9a Expression Vector Containing a Hybrid ospA/pspAGene

Specifically designed oligonucleotide primers were used in a PCRreaction to amplify the portion of the pspA gene of interest (in thiscase from amino acid 1 to 314) from the S. pneumoniae strain RX1.

The 5′-end primer had the nucleotide sequence: 5′-GGG ACA GCA TGC GAAGAA TCT CCC GTA GCC AGT-3′ (PspNl) SEQ ID NO: 1).

The 3′-end primer had the nucleotide sequence: 5′-GAT GGA TCC TTT TGGTGC AGG AGC TGG TTT-3′ (PspC370) (SEQ ID NO: 2).

The PCR reaction was as follows: 94° C. for 30 seconds to denature DNA;42° C. for one minute for annealing DNA; and 72° C. for one minute forextension of DNA. This was carried out for 25 cycles, followed by a 5minute extension at 72° C. This procedure introduced a stop codon atamino acid 315. The PCR product was purified using the Gene Clean IImethod (Bio101), and digested with SphI and BamHI.

The plasmid pLF100 was prepared as follows.

Plasmid pBluescript KS+ (Stratagene) was digested with XbaI and BamHIand ligated with a 900 bp XbaI-BamHI DNA fragment containing thecomplete coding region of B. burgdorferi strain ACA1 ospA gene, to forma lipoprotein fusion vector pLF100. This procedure is shownschematically in FIG. 1 of application Ser. No. 08/475,781, filed Jun.7, 1995 (abandoned; the continuation of which is application Ser. No.09/067,453, filed Apr. 28, 1998 (now U.S. Pat. No. 6,538,118 B1)) andincorporated herein by reference.

The vector pLF100 has been deposited with the American Type CultureCollection at Rockville, Md. on Feb. 2, 1995 under Accession No. 69750.This deposit was made under the terms of the Budapest Treaty.

pLF100 was digested with SphI and BamHI and the amplified pspA gene wasligated to this plasmid to form the plasmid pLF321, which contained thehybrid ospA-pspA gene. The hybrid gene was excised from pLF321 bydigestion with NdeI and BamHI and cloned into the NdeI and BamHI sitesof the plasmid vector pET9a to place the ospA-pspA hybrid gene under thecontrol of a T7 promoter. The resulting plasmid is called pPA321-L. Thisprocess is shown schematically in FIG. 9 of application Ser. No.08/475,781 filed Jun. 7, 1995 (abandoned; the continuation of which isapplication Ser. No. 09/067,453, filed Apr. 28, 1998 (now U.S. Pat. No.6,538,118 B1)) and incorporated herein by reference.

Example 2

Construction of a pET9a Expression Vector Containing the pspA Gene

Specifically designed oligonucleotide primers were used a PCR reactionto amplify the portion of the pspA gene of interest (in this case fromamino acid 1 to 314) from the S. pneumoniae strain RX1.

The 5′-end primer had the nucleotide sequence: 5′-GCT CCT GCA TAT GGAAGA ATC TCC CGT AGC C-3′ (PspNL-2) (SEQ ID NO: 3)

The 3′-end primer had the nucleotide sequence: 5′-GAT GGA TCC TTT TGGTGC AGG AGC TGG TTT-3′ (PspC370) (SEQ ID NO: 4).

The PCR reaction was as follows: 94° C. for 30 seconds to denature DNA;and 72° C. for one minute for annealing and extension of DNA. This wascarried out for 25 cycles, which was followed by a 5 minute extension at72° C. This procedure introduced a stop codon at amino acid 315. The PCRproduct was purified using the Gene Clean II method (Bio 101), anddigested with NdeI and BamHI. The digested PCR product was cloned intothe NdeI and BamHI sites of the plasmid vector pET9a to place the pspAgene under the control of a T7 promoter. The resulting plasmid is calledpPA321-NL. This process is shown schematically in FIG. 10 of applicationSer. No. 08/475,781, filed Jun. 7, 1995 (abandoned; the continuation ofwhich is application Ser. No. 09/067,453, filed Apr. 28, 1998 (now U.S.Pat. No. 6,538,118 B1)) and incorporated herein by reference.

Example 3

Expression and Purification of Lipidated PspA

Plasmid pPA321-L was used to transform E. coli strain BL21(DE3)pLyS. Thetransformed E. coli was inoculated into LB media containing 30 μg/mlkanamycin sulfate and 25 μg/ml chloramphenicol. The culture was grownovernight in a flask shaker at 37° C.

The following morning 50 ml of overnight culture was transferred to 1 LLB media containing 30 μg/ml kanamycin sulfate and the culture was grownin a flask shaker at 37° C. to a level of OD 600 nm of 0.6–1.0, inapproximately 3–5 hours. To the culture medium was added IPTG to a finalconcentration of 0.5 mM and the culture was grown for an additional twohours at 30° C. The cultures were harvested by centrifugation at 4° C.at 10,000×G and the cell pellet collected. Lipidated PspA was recoveredfrom the cell pellet.

The cell pellet was resuspended in PBS at 30 g wet cell paste per literPBS. The cell suspension was frozen and stored at −20° C. The cells werethawed to room temperature to effect lysis. DNase1 was added to thethawed material at a final concentration of 1 μg/ml and the mixtureincubated for 30 minutes at room temperature, which resulted in adecrease in viscosity of the material.

The material was then chilled in an ice bath to below 10° C. and Triton™X-114 was added as a 10% stock solution to a final concentration of 0.3to 1%. The mixture was kept on ice for 20 minutes. The chilled mixturewas then heated to 37° C. and held at that temperature for 10 minutes.This caused the solution to become very cloudy as phase separationoccurred. The mixture was then centrifuged at about 20° C. for 10minutes at 12,000×G, which caused a separation of the mixture into alower detergent phase, an upper clear aqueous phase and a pellet. Thelipidated PspA partitioned into the detergent phase. The detergent phasewas separated from the other two phases, diluted 1:10 with a buffercomprising 50 mM Tris, 2 mM EDTA, 10 mM NaCl pH 7.5, and was stored at−20° C.

A Q-Sepharose column was prepared in a volume of 1 ml per 5 ml diluteddetergent phase. The column was washed with 2 column volumes of a buffercomprising 50 mM Tris, 2 mM EDTA, 0.3% Triton™ X-100, 1M NaCl pH 4.0,and then equilibrated with 5 to 10 column volumes 50 mM Tris, 2 mM EDTA,0.3% Triton™ X-100, 10 mM NaCl pH 4.0. The pH of the diluted detergentphase material was adjusted to 4.0, at which time a precipitationoccurred. This material was passed through a 0.2 μM cellulose acetatefiltering unit to remove the precipitated material. The filtered diluteddetergent phase was applied to the Q-Sepharose column and the flowthrough (containing PA321-L) was collected. The column was washed with1–2 column volumes of 50 mM Tris, 2 mM EDTA, 0.3% Triton™ X-100, 10 mMNaCl pH 4.0, and the flow through was pooled with the previous flowthrough fraction. The pH of the flow through pool was adjusted to 7.5.The bound material, contaminating E. coli proteins, was eluted from theQ-Sepharose with 2 column volumes of 50 mM Tris, 2 mM EDTA, 0.3% Triton™X-100, 1M NaCl pH 4.0. A schematic of the purification process describedin this Example is shown in FIG. 11 of application Ser. No. 08/475,781,filed Jun. 7, 1995 (abandoned; the continuation of which is applicationSer. No. 09/067,453, filed Apr. 28, 1998 (now U.S. Pat. No. 6,538,118B1)) and incorporated herein by reference.

Example 4

Expression and Purification of Non-lipidated PspA

Plasmid pPA321-NL was used to transform E. coli strain BL21(DE3)pLyS.The transformed E. coli was inoculated into LB media containing 30 μg/mlkanamycin sulfate and 25 μg/ml chloramphenicol. The culture was grownovernight in a flask shaker at 37° C.

The following morning 50 ml of overnight culture was transferred to 1 LLB media containing 30 μg/ml kanamycin sulfate and the culture was grownin a flask shaker at 37° C. to a level of OD 600 nm of 0.6–1.0, inapproximately 3–5 hours. To the culture medium was added IPTG to a finalconcentration of 0.5 mM and the culture was grown for an additional twohours at 30° C. The cultures were harvested by centrifugation at 4° C.at 10,000×G and the cell pellet collected. Non-lipidated PspA wasrecovered from the cell pellet.

The cell pellet was resuspended in PBS at 30 g wet cell paste per literPBS. The cell suspension was frozen and stored at −20° C. The cells werethawed to room temperature to effect lysis. DNaseI was added to thethawed material at a final concentration of 1 μg/ml and the mixtureincubated for 30 minutes at room temperature, which resulted in adecrease in viscosity of the material. The mixture was centrifuged at 4°C. at 10,000×G, and the cell supernatant saved, which containednon-lipidated PspA. The pellet was washed with PBS, centrifuged at 4° C.at 10,000×G and the cell supernatant pooled with the previous cellsupernatant.

A MonoQ column (Pharmacia) was prepared in a volume of 1 ml per 2 mlcell supernatant. The column was washed with 2 column volumes of abuffer comprising 50 mM Tris, 2 mM EDTA, 1M NaCl pH 7.5, and thenequilibrated with 5 to 10 column volumes of a buffer comprising 50 mMTris, 2 mM EDTA, 10 mM NaCl pH 7.5. The cell supernatant pool wasapplied to the Q-Sepharose column and the flow through was collected.The column was washed with 2–5 column volumes of 50 mM Tris, 2 mM EDTA,10 mM NaCl pH 7.5, and the flow through pooled with the previous flowthrough.

The elution of bound proteins began with the first step of a 5–10 columnvolume wash with 50 mM Tris, 2 mM EDTA, 100 mM NaCl pH 7.5. The secondelution step was a 5–10 column volume wash with 50 mM Trie, 2 mM EDTA,200 mM NaCl pH 7.5. The non-lipidated PspA was contained in thisfraction. The remaining bound contaminating proteins were removed with50 mM Tris and 2 mM EDTA pH 7.5 with 300 mM-1M NaCl.

A schematic of the purification process described in this Example isshown in FIG. 12 of application Ser. No. 08/475,781, filed Jun. 7, 1995(abandoned; the continuation of which is application Ser. No.09/061,453, filed Apr. 28 1998 (now U.S. Pat No. 6,538,118 B1)) andincorporated herein by reference.

Example 5 Immunogenicity of Recombinant Lipidated PspA

Purified recombinant lipidated PspA, prepared as described in Example 3,was tested for immunogenicity in mice and compared to that fromnon-lipidated PspA prepared as described in Example 4. For this study,CBA/N mice were immunized subcutaneously in the back of the neck with0.5 ml of the following formulations at the indicated PspA antigenconcentrations.

PspA Antigen Formulation Concentration Native PspA molecule of the RX1strain 200 ng/ml (Native RX1) Non-Lipidated Recombinant PspA (pPA- 200and 1000 ng/ml 321-NL) Alone in PBS* Non-Lipidated Recombinant PspA(pPA- 200 and 1000 ng/ml 321-NL) Adsorbed to Alum Lipidated RecombinantPspA (pPA-321-L) 200 and 1000 ng/ml Alone in PBS Lipidated RecombinantPspA (pPA0321-NL) 200 and 1000 ng/ml Adsorbed to Alum* Alum* 0 ng/ml PBS0 ng/ml *Alum was Hydrogel at a concentration of 200 μg/ml

Four mice were immunized on days 0 and 21 for each dosage of theformulations. The mice were then bled on day 35 and subsequentlychallenged with S. pneumoniae of A66 strain. The days of survival afterchallenge for the mice were recorded and surviving mice were bled ondays 36, 37, 42 and 46. From these subsequent bleeds the blood wasassayed for the number of colony forming units (CFU) of S.pneumoniae/ml. The sera taken on day 35 were assayed by ELISA forantibodies against PspA using ELISA. The days to death for thechallenged mice are shown in the following table.

Survival in Immune and Non-Immune CBA/M Mice Immunization Efficacy doseDays to ρ value Alive: ρ value Group Antigen in μg Alum Death time todeath* Dead Survival* #1A pPA-321-L 1.0 − 4x > 14 0.01 4:0 0.01 #1BPpA-321-L 0.2 − 4x > 14 0.01 4:0 0.01 #2A pPA-321-L 1.0 + 4x > 14 0.014:0 0.01 #2B pPA-321-L 0.2 + 4x > 14 0.01 4:0 0.01 #3A pPA-321-NL 1.0 −1, 1, 2, 2 n.s. 0:4 n.s. #3B pPA-321-NL 0.2 − 1, 1, 2, ≧ 15 n.s. 1:3n.s. #4A pPA-321-NL 1.0 + 4x > 14 0.01 4:0 0.01 #4B pPA-321-NL 0.2 −4x > 14 0.01 4:0 0.01 #5    FL-Rx1 0.2 − 4x > H 0.01 4:0 0.01 #6    none0.0 + 1, 1, 3, 6 n.s. 0:4 n.s. #7    none 0.0 − 1, 1, 1, ≧ 15 n.s. 1:3n.s. pooled 0.0 5x1, 3, 6, ≧ 15 — 1:7 none Note: *indicates versuspooled controls; time to death, by one tailed two sample rank test;survival, by one tailed Fisher Exact test. Calculations have been doneusing “one tail” since we have never observed anti-PspA immunity toconsistently cause susceptibility.

The number of CFU in the blood of the nice are shown in the table below.

Bacteremia in Immune and Non-Immune CBA/M Mice Immunization Cog₁₀ CFUdose 1 2 6 7 Group Antigen in μg Alum day day day day #1A pPA-321-L 1.0− ≦1.6, 1.9, 2.1, 2.5 4x ≦ 1.6 4x ≦ 1.6 n.d. #1B pPA-321-L 0.2 − 3x ≦1.6, 1.7 4x ≦ 1.6 4x ≦ 1.6 n.d. #2A pPA-321-L 1.0 + 2x ≦ 1.6, 1.7, 2.93x ≦ 1.6, 1.7 4x ≦ 1.6 n.d. #2B pPA-321-L 0.2 + 2x ≦ 1.6, 1.7, 1.7 4x ≦1.6 4x ≦ 1.6 n.d. #3A pPA-321-NL 1.0 − ≦1.6, 1.7, d, d d, d, d, d d, d,d, d d, d, d, d #3B pPA-321-NL 0.2 − 2x > 7, d, d ≦1.6, d, d, d ≦1.6, d,d, d n.d., d, d, d #4A pPA-321-NL 1.0 + 2x ≦ 1.6, 6.7, >7 3x ≦ 1.6, 1.74x ≦ 1.6 n.d. #4B pPA-321-NL 0.2 + ≦1.6, 1.7, 2.1, 2.4 4x ≦ 1.6 4x ≦ 1.6n.d. #5    FL-Rx1 0.2 − 2x ≦ 1.6, 2.6, 2.7 4x ≦ 1.6 4x ≦ 1.6 n.d. #6   none 0.0 + ≦1.6, 4.1, >7, d ≦1.6, 5.1, d, d, 6.1, d, d, d d, d, d, d#7    none 0.0 − 1.7, >7, >7, d ≦I.6, d, d, d ≦1.6, d, d, d n.d, d, d, dpooled none 0.0 ≦1.6, 4.1, >7, >7, d 2x ≦ 1.6, 5.1, ≦1.6, 6.1 n.d, d, d,d, d, d, d d, d, d, d, d d, d, d, d, d Note: 1 colony at the highestconcentration of blood calculated out to 47 CFU or Log 1.7. Thus “1.6”indicates no colonies counted. >10⁷ indicates that the mouse was alivebut the number of colonies at the highest dilution was too high tocount, “d” indicates the nice had died prior to assay.

These results indicate that the recombinant protein was not protectivewhen injected alone. The recombinant antigen adjuvanted with alum and/orPAM₃cys lipidation was immunogenic and protective. The native fulllength PspA antigen did not need an adjuvant to be protective. The CFUresults indicate that mice protected by immunization cleared thechallenging S. pneumoniae from the blood in two days.

ELISA analysis of sera taken on day 35 indicated that there was a goodcorrelation between protection of the mice from S. pneumoniae challengeand the induction of measurable antibody responses. No detectableantibody responses were observed in the sera of mice immunized with thenon-lipidated antigen (pPA-321-NL) in saline or to the negative controlsthat did not contain PspA antigen, (as shown in the table below). Goodantibody responses were detected to the Native RX1 PspA antigen and tothe recombinant PspA when it was lipidated with PAM₃cys and/or adsorbedto alum.

ELISA Analysis of Day 35 Mouse Sera PspA PspA Alum Dose Resulting OD atIndicated Dilution of the Antisera* Antigen Adsorption (μg/mouse 6001200 2400 4800 9600 19200 pPA-321-L No 0.1 0.885 0.497 0.271 0.146 0.0750.039 (0.082) (0.043) (0.025) (0.017) (0.012) (0.009) pPA-321-L No 0.51.857 1.437 1.108 0.750 0.459 0.284 (0.060) (0.137) (0.150) (0.139)(0.092) (0.057) pPA-321-L Yes 0.1 1.373 1.048 0.745 0.490 0.288 0.171(0.325) (0.376) (0.362) (0.304) (0.197) (0.147) pPA-321-L Yes 0.5 1.2020.787 0.472 0.296 0.162 0.087 (0.162) (0.184) (0.187) (0.102) (0.061)(0.035) pPA-321-NL No 0.1 0.022 0.030 0.014 0.007 0.006 0.001 (0.035)(0.060) (0.024) (0.018) (0.005) (6.001) pPA-321-NL No 0.5 0.029 0.0140.008 0.003 0.002 0.002 (0.035) (0.014) (0.007) (0.004) (0.002) (0.002)pPA-321-NL Yes 0.1 0.822 0.481 0.278 0.154 0.082 0.042 (0.181) (0.166)(0.085) (0.051) (0.029) (0.015) pPA-321-NL Yes 0.5 1.017 0.709 0.4470.253 0.141 0.075 (0.139) (0.128) (0.101) (0.057) (0.034) (0.020) NativeRX1 No 0.1 1.367 1.207 0.922 0.608 0.375 0.209 (0.084) (0.060) (0.070)(0.077) (0.048) (0.029) None NO 0 0.018 0.012 0.009 0.005 0.005 0.005(0.003) (0.008) (0.003) (0.002) (0.002) (0.002) None Yes 0 0.013 0.0090.004 0.004 0.001 0.000 (0.006) (0.008) (0.004) (0.003) (0.001) (0.000)*The OD is the mean of the result of the four tested animals and thestandard deviation is in parentheses.

To determine whether protection was at least in part mediated by theanti-PspA antibody responses, a passive experiment was run. BALB/c micewere immunized with 0.5 μg of recombinant lipidated PspA alone orabsorbed to alum, or with recombinant non-lipidated PspA adsorbed toalum on days 0 and 21; and were bled on day 35. The anti-sera werediluted 1:3 or 1:15 in saline and 0.1 ml of the dilution was injectedi.p. into two mice for each dilution. A ⅓ dilution of normal BALB/cmouse serum was used as a negative control. Subsequently one hour afterpassive immunization, the animals were challenged i.v. with the WU2strain of S. pneumoniae (15,000 CFU). Mice passively immunized withanti-PspA sera were protected as compared to those mice that receiveddilutions of normal mouse sera as shown in the following table.

Passive Protection of BALB/c to WU2 Immunizing Formulation PepA PepADose Dilution Days to Daath Antigen Alum (μg/animal) of Serum PoetChallenge pAP-321-L No 0.5 3 4, >7 15  2, 4 pPA-321-L Yes 0.5 3 >7, >715  4, >7 pPA-321-NL Yes 0.5 3 2, 4 15  >7, >7 None No 0   3 2,2

Example 6

Combination PepA/Plu Vaccine

Purified recombinant lipidated PspA, prepared as described in Example 3,and non-lipidated PspA prepared as described in Example 4 were combinedwith split flu antigen from the A/Texas strain.

These combinations and the flu antigen alone were formulated either insaline or adsorbed to alum in saline. The alum when added was keptconstant at 100 μg/injection and the PspA was kept constant at 0.5μg/injection. The flu antigen was diluted to concentrations of 0.5, 0.1,0.02 and 0.004 μg/injection. Four BALB/c mice for each of theformulations were immunized on days 0 and 21, and were then bled on day35. The sera from the immunized mice were then assayed for their abilityto inhibit the agglutination or chicken red blood cells by A/Texas HAantigen. The resulting hemagglutination inhibition (HAI) titers areshown in the following table.

Flu Alum Flu HA Dose GMT of STD of GMT Antigen PspA Antigen Adsorption(μg/injection) HAI Titer of RAT Titer A/Texas — + 0.5 28.1 3 A/Texas — +0.1 21.8 6.6 A/Texas — + 0.02 22.8 52 A/Texas — + 0.004 16.1 3.8 A/Texas— − 0.5 12.4 5.3 A/Texas — − 0.1 23.8 3.3 A/Texas — − 0.02 19.2 2.8A/Texas — − 0.004 11.9 3.7 A/Texas pPA-321-L + 0.5 794.8 2.6 A/TexaspPA-321-L + 0.1 452.5 2.7 A/Texas pPA-321-L + 0.02 54.2 6.9 A/TexaspPA-321-L + 0.004 36.7 4.9 A/Texas pPA-321-L − 0.5 51.9 4 A/TexaspPA-321-L − 0.1 27.1 5.1 A/Texas pPA-321-L − 0.02 19.2 3.3 A/TexaspPA-321-L − 0.004 15.4 3.4 A/Texas pPA-321-NL + 0.5 174.5 2.7 A/TexaspPA-321-NL + 0.1 59.1 3.4 A/Texas pPA-321-NL + 0.02 19.2 5.1 A/TexaspPA-321-NL + 0.004 14.8 3.1 A/Texas pPA-321-NL − 0.5 35.1 2.7 A/TexaspPA-321-NL − 0.1 23.8 3 A/Texas pPA-321-NL − 0.02 14.8 2.9 A/TexaspPA-321-NL − 0.004 10.2 2.6 None None − 0 7.1 1.9

Example 7

Expression and Purification of Non-Lipidated OspC.

E. coli JM 109 transformants containing plasmid vector containingchromosomal gene fragment encoding non-lipidated OspC were prepared andgrown as described in WO 91/09870. The cultures were harvested, theculture medium centrifuged at 10,000×G for 10 minutes at 4° C., thesupernatant discarded and the pellet collected.

The cell pellet was first resuspended in lysis buffer A, namely 50 mMTris-HCl pH 8.0, 2 mM EDTA, 0.1 mM DTT, 5% glycerol and 0.4 mg/mllysozyme, and the suspension stirred for 20 minutes at room temperature.TRITON™ X-100 then was added to the cell suspension to a concentrationof 1 wt %, DNase I was added to a concentration of 1 μg/ml, and thesuspension stirred at room temperature for a further 20 minutes toeffect cell lysis. Sodium chloride next was added to the cell suspensionto a concentration of 1M and the suspension again stirred at 4° C. for afurther 20 minutes. The suspension then was centrifuged at 20,000×G for30 minutes, the resultant supernatant separated from the pellet and thepellet was discarded.

The separated supernatant was dialyzed against a buffer comprising 50 mMTris pH 8, 2 mM EDTA. The supernatant next was loaded onto aDEAE-Sepharose CL-6B column and the non-lipidated OspC was collected inthe column flow-through. The flow-through was dialyzed against a 0.1 Mphosphate buffer, pH 6.0.

The dialyzed flow-through next was bound to a S-Sepharose fast flowcolumn equilibrated with 0.1M phosphate buffer, pH 6.0. Purifiednon-lipidated OspC then was eluted from the S-Sepharose column using thedialysis buffer with 0.15 M NaCl added.

The aqueous solution of highly purified non-lipidated OspC was analyzedby Coomassie stained gels. The purity of the product was estimated to begreater than 80%.

Example 8

Potentiation of Response to Non-lipidated OspC with Lipidated OspA

Purified recombinant non-lipidated OspC, prepared as described inExample 7, was tested for immunogenicity in mice in combination with orwithout purified lipidated OspA (prepared as described in WO 92/14488).Formulations were administered with or without alum as an adjuvant. Theantigen dose tested in this experiment was 1 μg per dose. For thisstudy, 4 to 8 week old female C3H/He mice were immunized on day 0 andboosted on days 21 and 42.

Three representative animals were exsanguinated on days 21, 42, 63 and91. ELISA testing was performed on these sera using purifiednon-lipidated OspC as the coating antigen.

The only detectable OspC ELISA responses generated in this study werewith the formulation of OspC on alum. However, when lipidated OspA wasincluded on the alum the OspC ELISA response was 20-fold higher on day63 (as shown in FIG. 1) and 5-fold higher on day 91 (as shown in FIG.2). When lipidated OspA was included in the formulation without alumthere was no apparent effect on the immune response.

Example 9

Salivary Gland ELISPOT Analysis of Response to Urease with OspA

Mice (CH3/HeN; 4–5/group) were immunized by mucosal routes with theantigens indicated in the table below, on days 0 and 28. Proteins werediluted in PBS to a final volume of 25 μl for intranasal and 0.5 ml forintragastric. The mice were sacrificed for ELISPOTS at 15–17 days afterthe second immunization.

The ELISPOT protocol was derived from the one described by Mega et al.,J. Immunol. (1992), 148:2030–2039. The salivary glands were taken justafter sacrifice of the mouse, and placed immediately in a large volumeof RPMI 1640 medium (Gibco). The organs were cut in small pieces (1×1mm) using an automated tissue chopper (Mc Illwain tissue chopper, TheMickle Laboratory Engineering, Gilford, U.K.), and then digested in 2 mlof RPMI 1640 medium containing 5% FCS and 1 mg/ml of collagenase type IV(Sigma) for 30 minutes at 37° C. with gentle agitation. The digestedcells and fragments were passed through a 70 μM filter (Falcon), and thedigestion was repeated three more times. The digested cells were pooledand washed twice in a large volume of medium. The pooled cells were thenlysed using Gey's solution for 4 minutes on ice. After two more washes,the cells were resuspended in 2 ml of medium (+5% FCS), counted andaliquoted in 96 well nitrocellulose plates (MILLIPORE). The plates hadbeen coated overnight with 20 μg/ml of jackbean urease (BoehringerMannheim) or 10 μg/ml OspA (Connaught) in PBS at 4° C., and thensaturated with complete medium for 1 hour at 37° C. Two five-folddilutions of the cells were loaded in the wells (100 μl/well) inquadruplicate for each dilution and each isotype. After 4–16 hours at37° under 5% CO2, the cells were lysed 2×5 minutes in PBS/Tween 20(0.005%) and biotinylated anti-isotypes antibodies (Amersham) added fortwo hours at room temperature (dilution 1/1000). After 3 washes with PBSTween, biotinylated streptavidin peroxydase complex (Amersham) was addedfor 1 hour (dilution 1/500), and then spots revealed with3,9-aminoethylcarbazole (SIGMA). Once the plates dried, the spots werenumerated under a dissecting microscope (magnification 16 or 40×). Thevalues represent the means for 4 wells averaged for each group ofanimals.

As shown in the table below, it was found that lipidated OspAlipoprotein administered by mucosal routes without any added adjuvantinduced very strong local IgG and IgA responses, while non-lipidatedOspA did not induce any detectable responses. It was also found thatOspA had a powerful adjuvant effect on the local response to urease.

Anti Ure Anti Osp Spots/10⁶ Spots/10⁶ Cells Cells μg Jackbean Ure OspARoute IgA IgG IgA IgG — 1 L i.n. n.d. n.d.  583 345 — 1 NL i.n. n.d.n.d.   4  2 20 — i.n.  11  0 n.d. n.d. 20  1 L i.n. 189 18  742 257 2010 L i.n. 191 39 1237 174 20 + CT 10 μg — i.n. 478 42 n.d. n.d. 20 —i.n.,  0  1   25  0 i.g. 20 10 L i.n., 322 31 1919 177 i.g. i.n. =intransal i.n., i.g. = intransal & intragastric (the indicated dose wasgiven by each route) L = lipidated OspA; NL = non-lipidated OspA CT =cholera toxin (10 μg CTB + 10 ng CTX/mouse (PMSV)) n.d. = not determined

Example 10

ELISA Assay to Measure Serum Antibodies Against OspA and Urease

Mice (CH3/HeN; 4–5/group) were immunized by mucosal routes with theantigens indicated in the table in Example 9 on days 0 and 28. Proteinswere diluted in PBS to a final volume of 25 μl for intranasal and 0.5 mlfor intragastric. Blood was taken 9 days after the second immunization.

For the ELISA assay, flat-bottomed 96 well microliter plates (Dynatech)were coated with 100 μl/well of 1 μg/ml OspA (Connaught) or 2 μg/mljackbean urease (Boehringer Mannheim), diluted in 0.1 M sodium carbonatebuffer, pH 9.6. Plates were coated overnight at room temperature.

The following day, plates were washed 4× with PBS/0.05% Tween 20 andblocked with PBS with 1% BSA for 30 min. at room temperature. Afteranother wash, each well received 100 μl of PBS with 0.05% Tween 20 and0.1% BSA (PBS/T/B). Sera were pooled within each group of mice andserially diluted, and plates were incubated for 3 hr. at roomtemperature. After washing, 2° antibody biotinylated goat-antimouse IgGor IgA (Amersham) diluted 1:5,000 in PBS/T/B was added, and plates wereincubated for two hours at room temperature. Plates were washed againand incubated with streptavidin horseradish peroxidase (Amersham)diluted 1:2,000 in PBS/T/B for 1.5 hr. at room temperature. After afinal wash the substrate, OPD (Sigma), was added and plates wereincubated 10–20 min. Finally, the reaction was stopped with 50 μl 2 NH₂SO₄ and plates were read at 490/650 nm with a Molecular Devices platereader. The results shown in FIGS. 3, 4 and 5 demonstrate that lipidatedOspA, but not non-lipidated OspA, administered mucosally induces a verystrong serum IgG response. Additionally, lipidated OspA had a strongadjuvant effect on the serum IgG response to urease.

Having thus described in detail certain preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope thereof.

1. A method for inducing an immunological response in a host comprisingthe steps of: administering to the host at least one antigen, whereinthe at least one antigen is an influenza antigen; and administering tothe host a lipoprotein, wherein the lipoprotein is an expression productof a hybrid nucleic acid molecule, comprising a first nucleic acidsequence encoding a signal sequence of an OspA protein of Borrelia and asecond nucleic acid sequence encoding a S. pneumoniae PspA protein, orfragment of the PspA protein comprising amino acids 1 to 314 thereof,wherein the sequences are contiguous.
 2. The method of claim 1 whereinthe antigen and the lipoprotein are administered simultaneously.
 3. Themethod of claim 1 wherein, in the hybrid nucleic acid molecule, thefirst nucleic acid sequence and the second nucleic acid sequence arecoupled in a translational open reading frame relationship.
 4. Themethod of claim 1, wherein the antigen is an HA antigen.
 5. The methodof claim 1 wherein the lipoprotein is antigenic.
 6. The method of claim1 wherein the antigen and lipoprotein are administered mucosally.
 7. Themethod of claim 6 wherein the antigen and lipoprotein are administeredintranasally.
 8. The method of claim 6 wherein the antigen andlipoprotein are administered intragastrically.
 9. The method of claim 6wherein the antigen and lipoprotein are administered both intranasallyand intragastrically.
 10. The method of claim 1 wherein theimmunological response is therapeutic.
 11. The method of claim 1 whereinthe immunological response is prophylactic.